Herpes Simplex Virus
The herpes simplex virus (HSV) genome comprises two covalently linked segments, designated long (L) and short (S). Each segment contains a unique sequence flanked by a pair of inverted terminal repeat sequences. The long repeat (RL or RL) and the short repeat (RS or RS) are distinct.
The HSV ICP34.5 (also γ34.5) gene, which has been extensively studied1, 6, 7, 8, has been sequenced in HSV-1 strains F9 and syn17+3 and in HSV-2 strain HG524. One copy of the ICP34.5 gene is located within each of the RL repeat regions. Mutants inactivating both copies of the ICP34.5 gene (i.e. null mutants), e.g. HSV-1 strain 17 mutant 17162 (HSV 1716) or the mutants R3616 or R4009 in strain F5, are known to lack neurovirulence, i.e. be a virulent, and have utility as both gene delivery vectors or in the treatment of tumours by oncolysis. HSV-1 strain 17 mutant 1716 has a 759 bp deletion in each copy of the ICP34.5 gene located within the BamHI s restriction fragment of each RL repeat.
ICP34.5 null mutants such as HSV1716 are, in effect, first-generation oncolytic viruses. Most tumours exhibit individual characteristics and the ability of a broad spectrum first generation oncolytic virus to replicate in or provide an effective treatment for all tumour types is not guaranteed.
HSV 1716 is described in EP 0571410 and WO 92/13943 and has been deposited on 28 Jan. 1992 at the European Collection of Animal Cell Cultures, Vaccine Research and Production Laboratories, Public Health Laboratory Services, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom under accession number V92012803 in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (herein referred to as the ‘Budapest Treaty’).
Nitroreductase Prodrug Activation
Enzyme prodrug therapy is based on the enzymatic activation of a non toxic or low toxicity prodrug to a form that is considerably more cytotoxic. The activation may involve enzymatic reduction of the prodrug to a cytotoxic reduced form.
The E. coli nitroreductase enzyme (NTR) has been proposed for use in gene-directed enzyme prodrug therapy (GDEPT) as an activating enzyme for nitroaromatic prodrugs of the dinitrobenzamide class16. E. coli NTR is a homodimeric enzyme with two active sites and is the oxygen insensitive enzyme from E. coli (the nfsB gene product). It has the ability to reduce a wide range of nitro-containing compounds such as nitrofurazone (to the hydroxylamines) and quinones such as menadione (to the quinols). It is specifically inhibited by the irreversible inhibitor dicoumarol.
The ability of NTR to reduce aromatic nitro groups to the corresponding hydroxylamine (and possibly amine) derivatives has been proposed for cancer chemotherapy mainly with the dinitrobenzamide class of prodrugs. The 5-aziridin-1-yl-2,4-dinitrobenzamide CB1954 (CAS Registry number 21919-05-1) is one such prodrug which has been studied as a prodrug for GDEPT with NTR16.
Cyclic and acyclic nitroaryl phosphoroamide mustard analogues have also been shown to be activated by E. coli NTR17. The acyclic 4-nitrobenzyl phosphoramide mustard showed 167,500× selective cytotoxicity toward nitroreductase-expressing V79 cells with an IC50 as low as 0.4 nM which is about 100× more active and 27× more selective than CB1954.
Recombinant adenovirus and recombinant retrovirus10 expressing nitroreductase have been constructed for use with the prodrug CB1954 with the intention of providing a treatment for cancer. The recombinant virus is not oncolytic and relies on gene directed enzyme-prodrug therapy to achieve tumour cell kill.